JP2019030337A - Microorganism culture sheet - Google Patents

Abstract

To provide microorganism culture sheet, in which generation of warp is suppressed even in a case where cultivation layer containing polyvinyl pyrrolidone and in which is coloring reaction with coloring enzyme substrate is carried out in microorganism in broad range.SOLUTION: The present invention is microorganism culture sheet comprising a substrate sheet, a cultivation layer formed on the substrate sheet and a cover sheet covering the cultivation layer. The cultivation layer contains polyvinyl pyrrolidone, gelling agent, nutrition component, selective agent, coloring enzyme substrate and plasticizer. The coloring enzyme substrate is a coloring enzyme substrate degraded by β-galactosidase or degraded by α-glucosidase. The plasticizer is at least 1 selected from polyethylene glycol and 1,3-butylene glycol.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microorganism culture sheet, and preferably relates to a microorganism culture sheet used for detecting microorganisms.

  As a method for confirming the presence of microorganisms in a sample or measuring the number of microorganisms, there is an agar plate mixing method. In this agar plate mixing method, an agar medium formed in a pre-sterilized petri dish is used, so an autoclave for autoclaving the agar medium and an inspection room for performing microbiological examination aseptically are required. . In addition, operations such as sampling of microorganisms, preparation of sample solution, dispensing, and pour with a culture medium are required. Therefore, a microorganism culture sheet having a dry medium that can easily perform a microorganism test without requiring such a series of operations has been developed.

  For example, Patent Literature 1 describes a microorganism culture sheet having a base sheet, a culture layer formed on the base sheet, and a cover sheet that covers the culture layer. The culture sheet is formed by patterning a medium solution containing polyvinylpyrrolidone and at least one selected from the group consisting of a gelling agent, a nutritional component, a coloring indicator, a selection agent, and a substrate.

  Similarly, Patent Document 2 discloses a microorganism culture sheet comprising a base sheet, a culture layer provided on the top surface of the base sheet, and a cover film that can cover the culture layer. Yes.

International Publication No. 2011/007802 JP 2014-90701 A

  The microorganism culture sheets disclosed in Patent Documents 1 and 2 are excellent in work operability and safety, and work efficiency can be improved. In addition, since polyvinylpyrrolidone is an essential component of the medium solution, a culture layer having a predetermined shape can be formed in a cheap and simple pattern.

  However, when polyvinylpyrrolidone is used for the culture layer, the culture layer may be warped. If the culture layer is warped, the microorganism culture sheet is also warped, which leads to a decrease in work operability and safety. Therefore, in order to prevent the occurrence of such warpage, glycerin as a plasticizer is added to the culture layer. In the example of Patent Document 2, glycerin is also added.

  Here, conventionally, in order to detect coliforms, a color reaction test using β-galactosidase activity of the coliforms has been performed. That is, the coliform group grown on the culture layer decomposes a chromogenic enzyme substrate (hereinafter also referred to as β-galactosidase substrate) that is decomposed by β-galactosidase contained in the culture layer by β-galactosidase, thereby producing a chromogenic compound. Is released, and the colony develops color. However, as described above, in the culture layer using polyvinyl pyrrolidone, the culture layer is likely to warp. Therefore, the addition of glycerin as a plasticizer in the culture layer prevented the warpage from occurring. However, the present inventors surprisingly, when using a culture layer containing polyvinylpyrrolidone, glycerin, etc., among the Escherichia coli group originally having β-galactosidase activity, there is a bacterium that cannot confirm the color reaction by β-galactosidase substrate. I found it to be. Moreover, it has been found that such a phenomenon also occurs in a color development reaction test using α-glucosidase activity. Specifically, a color reaction test using α-glucosidase activity can be used for, for example, detection of Staphylococcus aureus, but when a culture layer containing polyvinylpyrrolidone, glycerin, or the like is used, It has been discovered that even in Staphylococcus aureus that normally should have a chromogenic reaction, there is a bacterium that cannot confirm the chromogenic reaction due to a chromogenic enzyme substrate that is degraded by α-glucosidase (hereinafter also referred to as α-glucosidase substrate). If there are bacteria that cannot achieve the desired color reaction, for example, when accurate information cannot be obtained in a test for measuring the number of living microorganisms, or in a test for detecting or identifying a target microorganism. In some cases, accurate results cannot be obtained.

  Accordingly, an object of the present invention is to provide a microorganism culture sheet that suppresses the occurrence of warping even when polyvinyl pyrrolidone is contained in the culture layer, and that allows a color development reaction with a chromogenic enzyme substrate to be obtained with a wide range of microorganisms. is there.

The present inventors have surprisingly found that by using polyethylene glycol or 1,3-butylene glycol instead of glycerin as a plasticizer, it is possible to obtain a color developing reaction with a β-galactosidase substrate and an α-glucosidase substrate. The present invention has been reached.
The present invention is expressed as follows.

(1) A microorganism culture sheet having a base sheet, a culture layer formed on the base sheet, and a cover sheet covering the culture layer,
The culture layer contains polyvinylpyrrolidone, a gelling agent, a nutrient component, a selective agent, a chromogenic enzyme substrate and a plasticizer,
The chromogenic enzyme substrate is a chromogenic enzyme substrate that is degraded by β-galactosidase or a chromogenic enzyme substrate that is degraded by α-glucosidase,
The microorganism culture sheet, wherein the plasticizer is at least one selected from polyethylene glycol and 1,3-butylene glycol.
(2) The microorganism culture sheet according to (1), wherein the plasticizer is polyethylene glycol.
(3) The microorganism culture sheet according to (2), wherein the polyethylene glycol has an average molecular weight of 100 or more and 1000 or less.
(4) The microorganism culture sheet according to (1), wherein the plasticizer is 1,3-butylene glycol.
(5) The microorganism culture sheet according to any one of (1) to (4), which is used to detect Escherichia coli, and the chromogenic enzyme substrate is a chromogenic enzyme substrate that is degraded by β-galactosidase.
(6) The microorganism culture sheet according to any one of (1) to (4), which is used to detect Staphylococcus aureus and the chromogenic enzyme substrate is a chromogenic enzyme substrate that is degraded by α-glucosidase.
(7) The microorganism culture sheet according to any one of (1) to (6), wherein at least one of the base sheet and the cover sheet is made of a transparent plastic sheet.

(8) A method for detecting Escherichia coli in a sample containing at least one kind of bacteria, wherein the sample is added to the culture layer of the microorganism culture sheet according to any one of (1) to (7). A step of inoculating at least a portion;
Culturing bacteria inoculated on the culture layer;
Including methods.
(9) A method for detecting Staphylococcus aureus in a sample containing at least one kind of bacteria,
Inoculating the culture layer of the microorganism culture sheet according to any one of (1) to (7) with at least a part of the sample;
Culturing bacteria inoculated on the culture layer;
Including methods.

  According to the configuration of the present invention, even when the culture layer contains polyvinyl pyrrolidone, it is possible to provide a microorganism culture sheet in which the occurrence of warpage is suppressed and a color development reaction with a chromogenic enzyme substrate can be obtained with a wide range of microorganisms.

It is a top view (a) and a sectional view (b) which show an example of a microorganism culture sheet, and is a figure showing a mode where a circular culture layer is formed in the approximate center of a substrate sheet. It is sectional drawing which shows an example of a microorganisms culture sheet, FIG.2 (a) is a figure which shows the aspect whose base material sheet is a multilayer structure, FIG.2 (b) is the aspect where a culture layer is a multilayer structure. FIG. It is the expanded view (a) and sectional drawing (b) which show an example of a microorganisms culture sheet, and is a figure which shows the aspect by which the culture component layer is formed in the base material sheet, and the specific component layer is formed in the cover sheet. It is the top view (a) and sectional view (b) which show an example of a microorganism culture sheet, and is a figure showing the mode by which the culture layer and the frame layer are formed on the substrate sheet. It is sectional drawing which shows an example of a microorganism culture sheet | seat, The aspect which is formed in the cover sheet so that a culture layer and a frame layer may be formed on a base material sheet, and a specific component layer may oppose the inner side of the said frame layer FIG. It is sectional drawing which shows an example of a microorganism culture sheet, a culture layer and a frame layer are formed on a base material sheet, a specific component layer faces the inner side of the said frame layer, and an adhesion layer is said frame layer It is a figure which shows the aspect currently formed in the cover sheet so that it may oppose. FIG. 7A is a cross-sectional view showing an example of a microorganism culture sheet in which a frame layer is formed on a base sheet, and FIG. 7A is a diagram showing an embodiment in which the base sheet has a multilayer structure. FIG. 7B is a diagram showing an embodiment in which the culture layer has a multilayer structure. It is a figure which shows an example of a microorganisms culture sheet, Comprising: The top view which shows the aspect by which the some culture layer by which the outer periphery was enclosed with the frame layer was formed on the base material sheet, and the cover sheet was fixed to the base material sheet It is (a) and sectional drawing (b). FIG. 2 is a diagram showing an example of a microorganism culture sheet, and is a development view (a) showing an aspect in which a culture layer having an outer periphery surrounded by a frame layer is formed on a base sheet on which a cover sheet is continuously provided; It is sectional drawing (b) and sectional drawing (c) which shows the aspect by which the said cover sheet | seat is bent and the culture | cultivation layer is coat | covered. It is a figure which shows an example of a microorganism culture sheet | seat, Comprising: It is sectional drawing which shows the aspect by which the cover sheet is fixed to the base material sheet in which the culture layer is formed with the double-sided adhesive tape. It is a figure which shows an example of a microorganisms culture sheet, Comprising: It is sectional drawing which shows the aspect by which the cover sheet is fixed to the base material sheet in which the culture layer and the frame layer are formed with the double-sided adhesive tape. It is an external appearance perspective view which shows an example of a microorganism culture sheet. It is an example of a microorganism culture sheet, and is an external perspective view in which a frame layer is formed on a base sheet. 2 is a photograph showing a state of a color development reaction on a culture layer after culturing in Example 1. FIG. 2 is a photograph showing a color development reaction on a culture layer after culturing in Example 2. FIG. 2 is a photograph showing a color development reaction on a culture layer after culturing in Comparative Example 1. FIG.

  The present invention is directed to a microorganism culture sheet having a base sheet, a culture layer formed on the base sheet, and a cover sheet that covers the culture layer. In the microorganism culture sheet of the present invention, the culture layer contains polyvinylpyrrolidone, a gelling agent, a nutrient component, a selective agent, a chromogenic enzyme substrate, and a plasticizer. Further, the chromogenic enzyme substrate is a chromogenic enzyme substrate that is degraded by β-galactosidase or a chromogenic enzyme substrate that is degraded by α-glucosidase. The plasticizer is at least one selected from polyethylene glycol and 1,3-butylene glycol.

  Hereinafter, embodiments of the present invention will be described. In addition, this invention is not limited to the following embodiment.

[Microbe culture sheet]
Hereinafter, each component of the microorganism culture sheet will be described.
(1) Culture layer In the microorganism culture sheet of the present invention, the culture layer provided on the base sheet contains polyvinylpyrrolidone, a gelling agent, a nutrient component, a selective agent, a chromogenic enzyme substrate, and a plasticizer. The culture layer can be formed from a medium solution containing a solvent for dispersing or dissolving these components.

(I) Polyvinyl pyrrolidone Polyvinyl pyrrolidone has a role as a viscosity modifier of a culture solution. According to polyvinylpyrrolidone, the viscosity of the medium solution can be easily adjusted by adjusting the concentration, so that the gelling agent, nutrient component, selective agent, chromogenic enzyme substrate and plasticizer can be uniformly dispersed. Moreover, it becomes possible to form a culture layer in a required part, and a waste of expensive material does not arise. Further, when the solvent is removed after the medium solution is arranged in a predetermined pattern, the culture layer can be formed by taking in the gelling agent and nutrient components because of the excellent film property of polyvinylpyrrolidone. Moreover, since it has favorable adhesiveness with the base material sheet, the culture layer can be formed without using an adhesive. Therefore, the influence of the growth of microorganisms due to the adhesive component can be reduced.

(Ii) Gelling agent Since a culture layer formed using a medium solution containing a gelling agent thickens or gels when inoculated with a test solution, it can give a good viscosity to the growth of microorganisms. it can. Moreover, since it can contact | adhere to a cover sheet after thickening, drying of the culture layer at the time of culture | cultivation can be suppressed.

  The gelling agent is not particularly limited, and a known gelling agent can be used. The gelling agent is preferably a high molecular polysaccharide, and examples thereof include carrageenan, guar gum, chitansan gum, hydroxyethyl cellulose, carboxymethyl cellulose, locust bean gum, alginic acid, and alginic acid salt. Since these high molecular polysaccharides are thickened or gelled by water contained in the test solution, the growth of microorganisms is improved. Moreover, since the high molecular polysaccharide is highly transparent, the visibility of colonies is excellent. Furthermore, since the polymer polysaccharide is thickened and easily adheres to the cover sheet, drying during culture can be suppressed. Of the high molecular polysaccharides, guar gum is particularly preferable. This is because guar gum exhibits an extremely high viscosity at a low concentration, so that an environment suitable for the growth of microorganisms can be created with a small amount. Guar gum is produced from the endosperm portion of guar seeds, which are annual legumes.

  It is preferable that content of a gelatinizer is 100 to 600 mass parts with respect to 100 mass parts of polyvinylpyrrolidone.

(Iii) Nutritional component The nutritional component is not particularly limited, and a known nutritional component can be used. As a nutrient component, for example, in the case of a microorganism culture sheet for inspection of general viable bacteria, the nutrient component includes a yeast extract / pepton / glucose mixture, a meat extract / peptone mixture, or a peptone / soybean peptone / glucose mixture, or the like. A mixture of dipotassium phosphate and / or sodium chloride is preferably used. In the case of a microorganism culture sheet for coliform group inspection, sodium desoxycholate, peptone, ammonium iron citrate, sodium chloride, dipotassium phosphate, lactose, peptone, lactose, dipotassium phosphate, etc. are preferably used as nutrient components. . In the case of a microbial culture sheet for S. aureus testing, nutritional components include meat extract, peptone, sodium chloride, mannitol, egg yolk mixture, or peptone, meat extract, yeast extract, sodium pyruvate, glycine, lithium chloride, A telluric acid egg yolk mixture or the like is preferably used. In the case of a microorganism culture sheet for vibrio testing, yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate, sodium chloride, beef bile, etc. are preferably used as nutrient components . In the case of a microorganism culture sheet for enterococci examination, bovine brain extract, heart extract, peptone, glucose, dipotassium phosphate, sodium nitride and the like are preferably used as nutrient components. In the case of a microorganism culture sheet for fungal examination, a peptone / glucose mixture, a yeast extract / glucose mixture, a potato extract / glucose mixture, or the like is preferably used as a nutrient component. One or more nutrient components can be selected and used according to the microorganism to be developed from these. In addition, when a nutrient component is insufficient in the culture layer, an insufficient nutrient component may be added to the test solution.

(Iv) Selection agent A culture layer contains the selection agent which suppresses growth of microorganisms other than the detection objective. As such a selective agent, for example, antibiotics such as antibiotics and synthetic antibacterial agents, dyes, surfactants, inorganic salts and the like are used. Examples of antibiotics include methicillin, ceftamethazole, cefixime, ceftazidime, cefsulosin, bacitracin, polymyxin B, rifampicin, novobiocin, colistin, lincomycin, chloramphenicol, tetracycline, or streptomycin. Examples of synthetic antibacterial agents include sulfa drugs, nalidixic acid, olaquindox, and the like. Examples of the pigment include crystal violet, brilliant green, malachite green, and methylene blue that have bacteriostatic or bactericidal action. Examples of the surfactant include Tergitol 7, dodecyl sulfate, lauryl sulfate, and the like. Examples of the inorganic salt include selenite, tellurite, sulfite, sodium nitride, lithium chloride, oxalate, and high-concentration sodium chloride. In addition to these, for example, taurocholate, glycine, bile powder, bile salt, or deoxycholate can be used.

In the case of a microorganism culture sheet for coliform group inspection, as a selective agent, bile salts, bile-derived substances such as deoxychol, surfactants such as tarditol 7 and sodium lauryl sulfate (SDS), pigments such as crystal violet, Alternatively, an antibiotic such as cephexime or novobiocin is preferably used. In the case of a microorganism culture sheet for S. aureus examination, as selective agents, lithium chloride (LiCl), sodium azide (NaN ₃ ), colistin, Vibrio bacteria growth inhibitory compound O / 129, aztreonam, amphotericin, colimycin, sodium chloride (NaCl) and at least one selected from deferoxamine are preferably used.

(V) Chromogenic enzyme substrate In the microorganism culture sheet of the present invention, as the chromogenic enzyme substrate, a chromogenic enzyme substrate (β-galactosidase substrate) that is degraded by β-galactosidase or a chromogenic enzyme substrate that is degraded by α-glucosidase (α- Glucosidase substrate). The chromogenic enzyme substrate may be a natural substrate or a synthetic substrate. The color development is desirably due to a variation in optical properties such as changes in absorption, fluorescence or emission. As the β-galactosidase substrate, any compound that is hydrolyzed by β-galactosidase activity and exhibits color development can be used.

  As the α-glucosidase substrate, any compound that is hydrolyzed by α-glucosidase activity and develops color can be used.

  Examples of chromogenic enzyme substrates include enzyme substrates based on indoxyl derivatives, enzyme substrates based on umbelliferone derivatives, enzyme substrates based on flavone derivatives, enzyme substrates based on alizarin derivatives, enzyme substrates based on naphthol benzein derivatives, nitrophenol derivatives Enzyme substrate based on naphthol derivative, enzyme substrate based on catechol derivative, enzyme substrate based on hydroxyquinoline derivative, enzyme substrate based on coumarin derivative, enzyme substrate based on resorufin derivative, enzyme substrate based on phenoxazine derivative, naphthol Examples include enzyme substrates based on derivatives and enzyme substrates based on naphthylamine derivatives. Among these, enzyme substrates based on indoxyl derivatives or enzyme substrates based on umbelliferone derivatives are preferred.

  Examples of the β-galactosidase substrate include 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 6-chloro-3-indolyl-β-D-galactopyranoside and orthonitrophenyl-β-D-galactopyrano Cid etc. are mentioned. Of these, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) is particularly preferred.

  Examples of the α-glucosidase substrate include 4-methylumbelliferyl-α-D-glucopyranoside, 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside, 5-bromo-6-chloro-3- Indolyl-α-D-glucopyranoside, 5-bromo-6-chloro-3-indolyl-α-D-glucopyranoside, 6-chloro-3-indolyl-α-D-glucopyranoside, 5-bromo-3-indolyl-α- D-glucopyranoside, o-nitrophenyl-α-D-glucopyranoside, phenyl-α-D-glucopyranoside, 3-nitrophenyl-α-D-glucopyranoside, 4-nitrophenyl-α-D-glucopyranoside, 3-indoxyl- α-glucopyranoside trihydrate, n-heptyl-α-D-glucopyranoside, 5- Romo-4-chloro-3-indoxyl-2-acetamido-2-deoxy-.alpha.-D-glucopyranoside, and the like. Of these, 4-methylumbelliferyl-α-D-glucopyranoside is particularly preferred.

  The content of the chromogenic enzyme substrate in the medium solution is, for example, 0.01 to 2.0 g / l. The content of the chromogenic enzyme substrate in the medium solution is preferably 0.02 to 0.2 g / l from the viewpoint of color contrast.

  The chromogenic enzyme substrate can be used in a wide pH range, for example, pH 5.5 to 10, preferably pH 6.5 to 10.

(Vi) Plasticizer In the microorganism culture sheet of the present invention, at least one selected from polyethylene glycol and 1,3-butylene glycol is used as the plasticizer.

  The average molecular weight of polyethylene glycol is preferably 100 or more, more preferably 150 or more, further preferably 200 or more, and particularly preferably 300 or more. The average molecular weight of polyethylene glycol is preferably 10,000 or less, more preferably 5000 or less, further preferably 2000 or less, particularly preferably 1000 or less, and preferably 800 or less. More particularly preferred.

  The content of polyethylene glycol in the culture layer is, for example, from 30 parts by weight to 90 parts by weight with respect to 100 parts by weight of polyvinylpyrrolidone. The content of polyethylene glycol in the culture layer is preferably 40 parts by mass or more and 80 parts by mass or less with respect to 100 parts by mass of polyvinylpyrrolidone from the viewpoint of suppressing warpage.

  The content of 1,3-butylene glycol in the culture layer is, for example, 30 parts by mass or more and 90 parts by mass or less with respect to 100 parts by mass of polyvinylpyrrolidone. The content of 1,3-butylene glycol in the culture layer is preferably 40 parts by mass or more and 80 parts by mass or less with respect to 100 parts by mass of polyvinylpyrrolidone from the viewpoint of suppressing warpage.

(Vii) Others The culture layer may contain a β-galactosidase expression inducer. Examples of the β-galactosidase expression inducer include isopropyl-1-thio-β-D-galactoside (hereinafter also referred to as IPTG) and methyl-β-D-thiogalactoside. It is known that IPTG strongly induces the synthesis of β-galactosidase of E. coli at a concentration of 0.05 to 0.1 mM (1.2 to 2.4 mg / 100 ml).

  The culture layer may contain a color development indicator. The coloring indicator can color colonies by reacting with specific substances produced by metabolism of microorganisms that grow during the culture process, recognizing pH changes, etc., and has the effect of making counting the number of colonies extremely easy. Have. Examples of the color development indicator include triphenyltetrazolium chloride (hereinafter referred to as TTC), tetrazolim salts such as p-tolyltetrazolium red, tetrazolium violet, veratryl tetrazolium blue, neutral red mixture, phenol red, bromthymol blue, or thymol blue. There are pH indicators such as mixtures.

  The culture layer may be composed of one layer or may be composed of two or more layers. For example, a first culture layer is formed with a medium solution containing a gelling agent (ii) in a polyvinylpyrrolidone solution (i), and a gelling agent (ii) and a nutrient component (iii) are added to the polyvinylpyrrolidone solution (i) thereon. ), A selective culture agent (iv), and a culture medium containing a chromogenic enzyme substrate (v), the second culture layer may be formed to be a culture layer used in the present invention.

  Moreover, depending on the components contained in the medium solution, a reaction may occur by mixing, and the mixing reaction can be prevented by containing these in another layer. Therefore, for example, when components A and B that cause a mixed reaction in the nutrient component are contained, two layers of a first culture layer from which the A component has been removed and a second culture layer from which the B component has been removed are laminated. It may be a culture layer.

(Viii) Formation of culture layer The culture layer can be formed by applying a medium solution in a predetermined pattern on a base sheet and drying it. By forming the culture layer in a pattern, the test liquid is naturally spread to a certain range by covering the cover sheet immediately after the test liquid is dropped, and thus excellent operability can be provided. In the present invention, “pattern formation” means that a medium solution is arranged on a base material sheet in a predetermined shape by printing, coating, coating, spraying or the like. The method for disposing the medium solution on the substrate is not particularly limited. Therefore, for pattern formation, screen printing, gravure printing, letterpress printing, transfer printing, flexographic printing, other printing, application by dispenser or inkjet, bar coating to be coated using a bar, knife coating, die coating, etc. Methods such as spraying such as coating and spraying can be used.

  The components contained in the medium solution may be added as a powder, or after dissolving these in a solvent, the medium solution may be mixed with the alcohol solution of polyvinyl pyrrolidone, for example. Depending on the type of coloring indicator, coloring may be conspicuous at the time of sterilization. Coloring may be performed using a sample solution containing a coloring indicator instead of being contained in the culture layer.

(2) Structure of microorganism culture sheet An example of a suitable aspect of the microorganism culture sheet is shown in FIGS. 1 (a) and 1 (b). 1A is a plan view, and FIG. 1B is a cross-sectional view taken along the line AA ′ in FIG.

  FIG. 1 shows a mode in which a culture layer 30 is formed at substantially the center of a square base sheet 10 and a square cover sheet 40 is provided so as to cover the culture layer 30. The culture layer 30 is patterned on the base material sheet 10. As shown in the cross-sectional view of FIG. 2A, the base sheet 10 may be a multilayer sheet composed of two layers of a base sheet 11 and a base sheet 13, and further, other than plastic. It may be a multilayer sheet in which layers are laminated. Moreover, as shown in FIG.2 (b), the culture layer 30 may be comprised by the multilayer of two or more layers.

  The shape of the culture layer 30 prepared by pattern formation is not limited to the circular shape shown in FIG. 1, but may be a square, a rectangle, other polygons, or an indeterminate shape.

  As shown in the development view of FIG. 3A, the microorganism culture sheet contains a culture layer 30 and other components such as a binder component and a nutrient component in both the base sheet 10 and the cover sheet 40. Each of the component layers 30 ′ may be formed. For example, when the gelling agent is contained in both the culture layer 30 and the specific component layer 30 ′, the specific component provided on the culture layer 30 on the base sheet 10 and the cover sheet 40. Since the layer 30 ′ is disposed to face the layer 30 ′, for example, when the culture layer 30 on the substrate sheet 10 is inoculated with the test solution and then covered with the cover sheet 40, the test solution Is absorbed by the culture layer 30 and the specific component layer 30 ′, and microorganisms can be observed in both the culture layer 30 and the specific component layer 30 ′. In addition, depending on the indicator, when it is contained in the culture layer 30, coloring may be conspicuous at the time of sterilization. In this case, the indicator is included in the specific component layer 30 ′ to prevent coloring at the time of sterilization. be able to. FIG. 3B is a cross-sectional view.

  Moreover, as shown in the plan view of FIG. 4A and the AA ′ line cross-sectional view of FIG. 4B, the microorganism culture sheet has a culture layer 30 formed at substantially the center of the base sheet 10, A circular frame layer 20 may be formed on the outer periphery of the culture layer. According to the frame layer 20, the water absorption range of the test liquid inoculated into the culture layer 30 can be more reliably limited. The shape of the frame layer 20 is not limited to a circle, and may be any of a square, an ellipse, a polygon, an indeterminate shape, etc., but the water absorption range of the test solution inoculated into the culture layer 30 is predetermined. It is preferable that a frame layer is formed so as to ensure a sufficient area. In this embodiment, as shown in the cross-sectional view of FIG. 5, a specific component layer 30 ′ may be further formed on the cover sheet 40 so as to face the culture layer 30 on the base sheet 10.

  Furthermore, as shown in FIG. 6, an adhesive layer 50 may be formed on the cover sheet 40 so as to face the frame layer 20. Since the frame layer 20 and the adhesive layer 50 are in close contact with each other, drying and contamination of the culture layer 30 can be more effectively prevented.

  In the microorganism culture sheet, as shown in the cross-sectional view of FIG. 7 (a), even when the frame layer 20 made of a hydrophobic resin is formed on the surface of the base sheet 10, the base sheet 10 The multilayer sheet which consists of two layers of the base material sheet 11 and the base material sheet 13 may be sufficient, and the multilayer sheet which laminates other layers other than a plastic may be sufficient. 7B, even if the frame layer 20 made of a hydrophobic resin is formed on the surface of the base sheet 10, the culture layer 30 is a multilayer of two or more layers. It may be comprised.

  A plurality of culture layers 30 may exist on the base sheet 10. A mode in which a plurality of culture layers 30, a frame layer 20 made of a convex hydrophobic resin surrounding the outer periphery thereof, and a cover sheet 40 covering the culture layer 30 are fixed at the end of the base material sheet 10. Is shown in FIGS. 8 (a) and 8 (b). FIG. 8A is a plan view, and FIG. 8B is a cross-sectional view taken along line A-A ′ of FIG. In FIG. 8 (b), the cover sheet 40 is in a form in which two corners (C1, C2) are formed so as to cover the culture layer 30 and be in close contact with the upper part of the frame layer 20. ing. Although different from FIG. 8, a specific component layer 30 ′ may be formed on the cover sheet 40 so as to face the culture layer 30 formed on the substrate sheet 10.

(3) Substrate sheet In the microorganism culture sheet of the present invention, since the medium solution is patterned on the substrate sheet, the substrate sheet needs to have solvent resistance and printability. Moreover, as a microorganism culture sheet, water resistance is also required, and it is also required to have heat resistance that can withstand a drying treatment when forming a culture layer. The base sheet may be a single layer or a laminated sheet having two or more layers.

(4) Cover sheet In the microorganism culture sheet of the present invention, the cover sheet is preferably fixed to the base sheet. The cover sheet has the effect of preventing contamination by falling bacteria during culture and preventing moisture evaporation of the culture layer. It is preferable that the cover sheet is waterproof and water vapor impermeable, and at the same time transparent so that colonies can be observed and counted through the cover sheet after culturing the microorganisms. As the cover sheet, for example, a polyolefin sheet such as polyethylene and polypropylene as exemplified in the base sheet, a plastic sheet such as polyester, polyamide, polystyrene, polycarbonate, and polyvinyl chloride can be used. In the microorganism culture sheet, if the substrate sheet and the cover film are transparent, they can be observed with the projection light from the back surface of the microorganism culture sheet, and can also be observed with the incident light from the side surface. That is, since colonies can be observed and counted from both the cover sheet side and the base sheet side, the range of selection of the observation method and the measurement method is widened. In view of workability such as opening and closing of the cover sheet, a polyester film and a polyolefin film are particularly preferable.

(5) Frame layer In the microorganism culture sheet of the present invention, it is preferable that a frame layer made of a hydrophobic resin is formed on the outer periphery of the culture layer. When the frame layer is formed, when the test solution is inoculated into the culture layer and covered with a cover sheet, the test solution is quickly diffused to the frame layer. The cover sheet can be covered without waiting, and the inoculation operation can be performed in a short time. In addition, leakage of the test liquid can be prevented more reliably by the frame layer, and the work efficiency at the time of inoculation can be improved. When the culture layer contains a gelling agent, the test solution is absorbed by the culture layer inside the frame layer, the gelling agent thickens, and the adhesion between the culture layer and the cover sheet causes the culture layer to Drying can be prevented. Moreover, even when the gelling agent is not contained in the culture layer, since the polyvinyl pyrrolidone dissolves and thickens, it is possible to prevent the culture layer from drying due to the adhesion between the culture layer and the cover sheet. When the adhesion between the culture layer and the cover sheet is excellent, it is not necessary to form a layer for colony formation on the cover sheet, and excellent visibility of the colonies can be ensured.

(6) Adhesive layer In the microorganism culture sheet of this invention, an adhesive layer can be formed in a base material sheet or a cover sheet. Such an adhesive layer is disposed for the purpose of fixing the cover sheet covering the culture layer and preventing moisture evaporation and contamination of the culture layer, and any kind of adhesive may be used as long as the base sheet can be sealed. . In consideration of workability, a pressure-sensitive adhesive having a weak adhesive strength is preferable to the extent that the base sheet and the cover sheet can be re-bonded. For example, a rubber adhesive and an acrylic adhesive are preferable, and a re-peeling type and a slightly adhesive type acrylic adhesive are particularly preferably used. Specifically, a monomer containing a functional group such as a carboxyl group, a hydroxyl group, or an amino group with respect to 100 parts by mass of an alkyl acrylate such as butyl acrylate or 2-ethylhexyl acrylate (preferably an alkyl having 2 to 12 carbon atoms). A rosin, xylene resin, phenol resin, or the like as a tackifier may be added to a copolymer obtained by copolymerization with 2 to 15 parts by mass and crosslinked with melamines, isocyanates, epoxies, or the like. The pressure-sensitive adhesive layer may have any shape, but it is preferable that the adhesive layer is disposed so that the adhesive portion between the base sheet and the cover sheet can be bonded without a gap.

(7) Lattice Print Layer A lattice print layer may be laminated on the microorganism culture sheet of the present invention. For the grid printing, gravure printing or the like is preferable because it has a wide selection range of colorant, resin, solvent and the like. The size of the mesh is about 1 cm square.

(8) Microorganism culture kit In the present invention, a microorganism culture kit can be obtained by combining the microorganism culture sheet and a test solution containing a specific component.
A component (for example, a color indicator) that is desirably added to the culture layer can be added to the test solution and cultured. It is particularly effective when it contains components that are susceptible to alteration, discoloration, decomposition, etc. during sterilization and other processes.

(9) Production method (i) Formation of culture layer As described above, the culture layer in the microorganism culture sheet of the present invention is formed by placing a medium solution in a predetermined pattern on a substrate sheet and drying it. be able to. Also, the pattern formation method is not particularly limited as long as the medium solution can be applied to the predetermined shape of the base material sheet, and any method such as printing, application, coating, spraying and the like can be used. Preferably, after preparing a medium solution containing a gelling agent, a nutrient component, a selective agent, a chromogenic enzyme substrate and a plasticizer in an alcohol solution of polyvinylpyrrolidone, and patterning the medium solution on a base sheet The alcohol contained in the medium solution is removed to form a culture layer.

  In the method for producing a microorganism culture sheet, it is preferable to use an alcohol solution of polyvinyl pyrrolidone for reasons such as adhesion between the base sheet and the culture layer and ease of pattern formation. As alcohol, a C1-C5 alcohol can be used conveniently. For example, at least one selected from methanol, ethanol, isopropyl alcohol, and butanol can be used. Among these, methanol, ethanol, or isopropyl alcohol is preferable. Conventionally, high-boiling solvents such as water and toluene have been used to dissolve nutrients, but it is necessary to heat at a high temperature to remove the solvent, and pyrolysis of the components and substrate contained in the culture layer There was a risk of inviting. In addition, it is necessary to use excessive heat energy for solvent removal, which contributes to an increase in cost, and also causes a decrease in production efficiency because the solvent removal time becomes longer. In this embodiment, polyvinyl pyrrolidone is used, and the alcohol having a low boiling point is used as a solvent thereof, thereby avoiding thermal denaturation of components and base materials contained in the medium solution and improving production efficiency. Can do. It should be noted that a solvent in which an alcohol and a secondary solvent such as chloroform are mixed can also be used within a range not impairing the above-mentioned purpose.

  The reason why polyvinylpyrrolidone is an essential component of the medium solution is that the gelling agent, nutrient component, selective agent, chromogenic enzyme substrate and plasticizer can be uniformly dispersed by adjusting the concentration as described above. Moreover, the viscosity can be easily adjusted. Thereby, pattern formation of a culture solution can be performed easily. Polyvinylpyrrolidone has excellent film-forming properties and adhesion to the substrate sheet, so that a culture layer can be formed on the substrate sheet without using an adhesive. Can be avoided.

  The amount of alcohol relative to polyvinylpyrrolidone is preferably 100 parts by mass or more and 10000 parts by mass or less, more preferably 300 parts by mass or more and 2000 parts by mass or less, and more preferably 900 parts by mass or more with respect to 100 parts by mass of polyvinylpyrrolidone. The amount is particularly preferably 1400 parts by mass or less.

  The gelling agent constituting the medium solution need not be dissolved in an alcohol solution of polyvinylpyrrolidone as long as pattern formation is possible. Therefore, in the method for producing a microorganism culture sheet, the gelling agent can be added as a powder. When added as a powder, the gelling agent has an average particle size of 5 to 500 μm, more preferably 10 to 200 μm. When the average particle diameter is in the above range, the dispersibility is excellent. If it exceeds 500 μm, the dispersibility may be lowered, and the pattern formation suitability may be impaired. However, these may be dissolved in a solvent and mixed with the polyvinylpyrrolidone alcohol solution to form a medium solution.

  The content of the gelling agent in the culture medium is preferably 100 parts by mass or more and 600 parts by mass or less, and more preferably 250 parts by mass or more and 400 parts by mass or less with respect to 100 parts by mass of polyvinylpyrrolidone. If the content of the gelling agent is in the above range, it can be thickened or gelled by the water under the test droplet, and can have a good viscosity for growing bacteria.

  In addition, since the polyvinyl pyrrolidone film may be hard and brittle, in the present invention, polyethylene glycol or 1,3-butylene glycol is added as a plasticizer. In the present invention, an ether ester derivative plasticizer, a propylene glycol or glycol derivative plasticizer, a glycol ether derivative plasticizer, a polyhydroxycarboxylic acid derivative plasticizer, or a phthalic acid derivative system unless the effect of the present invention is inhibited. You may add other plasticizers, such as a plasticizer. It is desirable not to contain glycerin or a glycerin derivative plasticizer.

  In the method for producing a microorganism culture sheet, the medium solution is used to form a pattern. In particular, the medium solution can be used to suitably form a culture layer by coating with a dispenser or the like.

  As described above, the culture layer may be a multilayer of two or more layers. In the present invention, when the culture layer is a two-layer composed of the first culture layer and the second culture layer, pattern formation may be performed by different methods. For example, the first culture layer may be applied with a dispenser to form a pattern, and other methods such as spraying may be used for pattern formation of the second culture layer.

  In the present embodiment, after the medium solution is patterned on the base sheet, alcohol contained in the medium solution is removed. By using a culture layer in which a gelling agent or the like is blended with polyvinylpyrrolidone, microbial colonies can be observed and counted in the same manner as the agar medium, and extremely excellent visibility can be obtained. In addition, since a gelatinizer is water-soluble, it is common to apply to a base material, after preparing as aqueous solution. However, the gelling agent needs to be subjected to heat treatment for a long time at a high temperature in order to remove water, and bubbles and the like may be mixed due to high viscosity when preparing an aqueous solution. According to this embodiment, since powders such as gelling agents are dispersed in an alcohol solution of polyvinyl pyrrolidone, air bubbles can be avoided and water is not used, so drying is performed at a low temperature and in a short time. be able to. Further, since the gelling agent or the like is only dispersed in the polyvinylpyrrolidone alcohol solution but not dissolved, it is possible to prevent an increase in the viscosity of the medium solution, thereby enabling smooth pattern formation. . Moreover, in the obtained culture layer, since the above components are coated with polyvinyl pyrrolidone, when the test solution is inoculated, polyvinyl pyrrolidone dissolves immediately and the above components dissolve or absorb water. Therefore, it can be covered with a cover sheet immediately after inoculation with the test solution. Also, when polyvinylpyrrolidone dissolves or contains a gelling agent, it thickens and adheres closely to the cover sheet, thus preventing drying and ensuring extremely excellent visibility. Can do.

  In the microorganism culture sheet, the specific component layer may be applied, for example, on the entire surface or pattern formation. In the case of pattern formation, printing, application, coating, spraying, and the like may be performed as in the case of forming the culture layer.

  The culture layer only needs to be able to remove the alcohol contained in the medium solution, and can be dried according to conventionally known methods such as temperature and pressure.

The thickness of the culture layer formed on the base sheet is preferably 50 to 1000 μm, more preferably 200 to 600 μm after drying. Moreover, it is preferable that it is 5-400 g / m < ² > after drying, and, as for an application quantity, it is more preferable that it is 100-300 g / m < ² >. Within the above range, the culture layer can have a viscosity that is optimal for the growth of bacteria.

(Ii) Formation of frame layer The microorganism culture sheet of the present invention may form a frame layer on the surface of the base material sheet and on the outer periphery of the culture layer. The frame layer can be formed using a hydrophobic resin. In addition, the manufacturing method in particular of a frame layer is not ask | required. A base sheet in which a frame layer is formed in advance can also be used.

  The base sheet on which the frame layer is formed is a base sheet having at least a hydrophobic resin on the surface, and a convex portion is formed in the shape of the frame by embossing the hydrophobic resin portion. Can be mentioned. And if a culture layer is formed inside the said convex part, a microorganisms culture sheet can be manufactured. Moreover, the location which forms a culture layer can be embossed in concave shape, and the convex part formed in the outer periphery of a recessed part can also be used as a frame layer.

(Iii) Fixing of the cover sheet The microorganism culture sheet of the present invention covers the culture layer with the cover sheet after inoculating the test solution on the culture layer and cultures it, so that a part of the cover sheet is a base sheet. It is preferable that it is fixed to. However, in the case of a direct stamp, a wiping test, or the like, if the work efficiency is improved by the absence of the cover sheet, the cover sheet may be removed and used.

  When the cover sheet and the base sheet are composed of the same member, the cover sheet prepared separately may be fixed to the base sheet by heat sealing, laminating, or the like. The same material is used for the sheet, and the base sheet 10 and the cover sheet 40 are continuously connected to the base sheet 10 as shown in the developed view of FIG. 9A and the cross-sectional view of FIG. The microbial culture sheet may be formed by forming the frame layer 20 and the culture layer 30 and bending the cover sheet 40. FIG. 9C shows a cross-sectional view thereof. According to this method, even when the specific component layer 30 ′ or the adhesive layer 50 is formed on the cover sheet, the base material sheet 10 and the cover sheet 40 are continuously provided, and therefore the culture layer 30 on the base material sheet 10. The specific component layer 30 ′ can be formed accurately and easily at a position facing the surface.

  Moreover, as shown in the cross-sectional views of FIGS. 10 and 11, the cover sheet 40 and the base sheet 10 are made of different members, and the cover sheet 40 and the base sheet 10 on which the culture layer 30 is formed. You may stick together with the double-sided adhesive tape 60 grade | etc.,.

  12 and 13 are external perspective views of the microorganism culture sheet in which grid printing is formed on the cover sheet 40 and the base sheet 10 on which the cover sheet 40 and the culture layer 30 are formed is bonded at the end. In FIG. 13, the frame layer 20 is formed around the culture layer 30.

  The microorganism culture sheet can be sterilized by gamma ray irradiation or ethylene oxide gas sterilization to obtain a product of the microorganism culture sheet. The microorganism culture sheet can cultivate various microorganisms according to the type of microbial nutrients contained, the base sheet used, the air permeability of the cover sheet, and the like.

  Moreover, coliform bacteria contained in a sample can be effectively detected using the microorganism culture sheet of the present invention. That is, the microorganism culture sheet of the present invention has an excellent detection performance for coliforms in a color development reaction by β-galactosidase activity, and thus is useful as a tool for detecting coliforms. Similarly, Staphylococcus aureus contained in a sample can be effectively detected using the microorganism culture sheet of the present invention. That is, since the microorganism culture sheet of the present invention has an excellent detection performance for Staphylococcus aureus in the color development reaction by α-glucosidase activity, it is useful as a tool for detecting Staphylococcus aureus.

  Therefore, one aspect of the present invention is a method for detecting coliform bacteria in a sample containing at least one kind of bacteria, wherein at least a part of the sample is added to the culture layer of the microorganism culture sheet of the present invention. The method includes a step of inoculating and a step of culturing bacteria inoculated on the culture layer.

  One embodiment of the present invention is a method for detecting Staphylococcus aureus in a sample containing at least one kind of bacteria, wherein the culture layer of the microorganism culture sheet of the present invention includes at least a part of the sample. And a step of culturing bacteria inoculated on the culture layer.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

Example 1
24 g of polyvinyl pyrrolidone was dissolved in 276 g of methanol to prepare a methanol solution of polyvinyl pyrrolidone. In this methanol solution of polyvinyl pyrrolidone, 75.8 g of guar gum powder as a gelling agent, 9.3 g of tryptone as a nutritional component, 0.9 g of yeast extract, 0.35 g of sodium pyruvate, 0.35 g of L-tryptophan, sodium chloride 1.7 g, sodium dihydrogen phosphate 0.18 g, disodium hydrogen phosphate 0.95 g, potassium nitrate 0.18 g, sodium lauryl sulfate 0.04 g as a selective agent, X-gal 0.04 g as a chromogenic enzyme substrate, plasticity A medium solution was prepared by adding 10 g of polyethylene glycol as an agent and 0.04 g of IPTG as a β-galactosidase expression inducer. In addition, the acquisition place of the used material is as follows.

・ Polyvinylpyrrolidone (made by Nippon Shokubai Co., Ltd., trade name “Polyvinylpyrrolidone K-90”) ・ Guar gum powder (made by Sanki Co., Ltd., trade name “Neovisco G”, 200 mesh type)
・ Tryptone (Becton, Dickinson and Company, trade name “BACT TRYPTONE”)
-Yeast extract (Becton, Dickinson and Company, trade name “YEAST EXTRACT”)
・ Sodium pyruvate (Wako Pure Chemical Industries)
・ L-tryptophan (Wako Pure Chemical Industries)
・ Sodium chloride (Wako Pure Chemical Industries)
・ Sodium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd.)
・ Disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Potassium nitrate (Wako Pure Chemical Industries, Ltd.)
・ Sodium lauryl sulfate (Wako Pure Chemical Industries, Ltd.)
X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, manufactured by BIOSYTHH)
・ Polyethylene glycol (Wako Pure Chemical Industries, polyethylene glycol 600, Wako first grade)
・ IPTG (Wako Pure Chemical Industries)

Next, this medium solution was placed in a predetermined pattern on a polyethylene terephthalate film (trade name “Teijin Tetron Film” manufactured by Teijin DuPont), and then dried at 50 ° C. for 15 minutes to form a culture layer. The thickness of the culture layer after drying was about 250 μm. Next, using a dispenser, UV curable ink (manufactured by Jujo Chemical Co., Ltd., trade name “Reicure GA 4100-2”) was applied in a circle having a width of 1.0 mm, a height of 750 μm, and a diameter of 52 mm. Subsequently, an ultraviolet ray having an accumulated light amount of 250 mJ / cm ² was irradiated using a mercury lamp to cure the UV curable ink, and a frame layer was formed using a hydrophobic resin around the culture layer.

  And, after laminating a cover sheet (manufactured by Tosero Co., Ltd., OPP film, model number “OP U-1 (single-sided corona discharge treatment)”, thickness: 40 μm) on the polyethylene terephthalate film and bonding the ends, Sterilized with gamma rays to prepare a microorganism culture sheet (1).

(Example 2)
A microorganism culture sheet (2) was prepared in the same manner as in Example 1 except that 10 g of 1,3-butylene glycol (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of 10 g of polyethylene glycol. .

(Comparative Example 1)
A microorganism culture sheet (3) was produced in the same manner as in Example 1, except that 10 g of glycerin (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was used instead of 10 g of polyethylene glycol.

"Evaluation"
Escherichia coli (ATCC 25922) or Klebsiella pneumoniae (ATCC 13883) (both included in E. coli group) were cultured with shaking in a liquid medium (TRYPTIC SOY BROTH) at 37 ° C. for 24 hours. Then, each of the obtained test solutions was diluted with sterile physiological saline so that the number of bacteria was 10 ² / ml, and TTC was added to prepare two test solutions for culture test. 1 ml of each of these test liquids for culture test is inoculated on the culture layers of the microorganism culture sheets (1) to (3), and a cover sheet is placed on each of the culture layers. Spread out. Then, it left still for about 1 minute, the gel was formed, and six samples for culture tests were obtained. The obtained sample for culture test was cultured at 37 ° C. for 48 hours. And the presence or absence of color development was confirmed about the colony which generate | occur | produced after culture | cultivation. Photographs of culture test samples after culture are shown in FIGS. 14 shows a sample for culture test using the microorganism culture sheet (1) prepared in Example 1, and FIG. A culture test sample inoculated with a test solution of E. coli, FIG. The sample for culture | cultivation tests which inoculated the test liquid of pneumoniae is shown. FIG. 15 shows a culture test sample using the microorganism culture sheet (2) prepared in Example 2, and FIG. A sample for a culture test inoculated with a test solution of E. coli, FIG. The sample for culture | cultivation tests which inoculated the test liquid of pneumoniae is shown. FIG. 16 shows a sample for culture test using the microorganism culture sheet (3) prepared in Comparative Example 1, and FIG. A sample for culture test inoculated with a test solution of E. coli, FIG. The sample for culture | cultivation tests which inoculated the test liquid of pneumoniae is shown.

  In the microorganism culture sheet (1) or (2) (Examples 1 and 2) containing polyethylene glycol or 1,3-butylene glycol as a plasticizer, as shown in FIG. coli (ATCC 25922) and K.I. In both pneumoniae (ATCC 13883), color development by X-Gal was confirmed. However, in the microorganism culture sheet (3) (Comparative Example 1) containing glycerin as a plasticizer, as shown in FIG. Although color development is confirmed in E. coli (ATCC 25922), K. No color development was confirmed with pneumoniae (ATCC 13883). From the results of Comparative Example 1, it can be seen that when glycerin is used, there are bacteria in the E. coli group that cannot confirm the coloring reaction by the β-galactosidase substrate. Surprisingly, by using polyethylene glycol or 1,3-butylene glycol as a plasticizer instead of glycerin, even when the glycerin is used, color development reaction with β-galactosidase substrate is not possible even when the color is confirmed. You can see that.

  In the following test examples, in the color reaction test using α-glucosidase activity or phosphatase activity, there is a bacterium that does not produce a color reaction when using a culture layer containing glycerin, and polyethylene glycol instead of glycerin. Whether or not a color development reaction was obtained when using was examined in a simple manner on an agar medium.

(Reference Example 1)
A medium solution containing tryptone 4.0 g / l, polyethylene glycol 18.7 g / l, 4-methylumbelliferyl-α-D-glucopyranoside 0.1 g / l, and agar 15.0 g / l was prepared. . This medium solution was sterilized by an autoclave and then spread and hardened in a petri dish to prepare an agar medium (1).

(Reference Example 2)
An agar medium (2) was prepared in the same manner as in Reference Example 1 except that polyethylene glycol was not added to the agar medium solution.

(Reference Example 3)
Instead of 0.1 g / l 4-methylumbelliferyl-α-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-phosphate, a chromogenic enzyme substrate for phosphatase, at a concentration of 0.1 g / l In the same manner as in Reference Example 1 except that glycerin was added to the agar medium at a concentration of 18.7 g / l instead of polyethylene glycol 18.7 g / l. 3) was produced.

(Comparative Example 2)
An agar medium (4) was prepared in the same manner as in Reference Example 1, except that glycerin was added to the agar medium solution at a concentration of 18.7 g / l instead of polyethylene glycol 18.7 g / l.

"Evaluation"
As a bacterium having α-glucosidase activity, aureus (ATCC 25923), S.A. aureus (ATCC 25904), and S. aureus. aureus (ATCC 13276) was used. These three types of bacteria were cultured with shaking in a liquid medium (TRYPTIC SOY BROTH) at 37 ° C. for 24 hours. Then, the obtained test solution was diluted with sterilized physiological saline so that the number of bacteria was 10 ² / ml, and three test solutions for culture test were prepared. Each 0.1 ml of this test liquid for culture test was inoculated on each agar medium (1) to (4) and smeared using a conage bar to obtain a sample for culture test. The obtained sample for culture test was cultured at 35 ° C. for 48 hours. Table 1 shows the results of the color development reaction. In Table 1, o indicates that a color development reaction with an α-glucosidase substrate or a phosphatase substrate was confirmed. X indicates that the color development reaction by the α-glucosidase substrate was not confirmed.

  In Comparative Example 2, when glycerin was included as a plasticizer, a color reaction by the α-glucosidase substrate was not confirmed. Further, in Reference Example 2 in which no plasticizer was added, a coloring reaction with an α-glucosidase substrate was confirmed. As a result, it can be seen that in bacteria that can usually be detected by a color reaction with an α-glucosidase substrate, there are bacteria in which the color reaction cannot be confirmed due to the presence of glycerin. Moreover, in Reference Example 1 using polyethylene glycol as the plasticizer, it was possible to confirm the color development reaction with the α-glucosidase substrate for all the tested bacteria. In addition, from Reference Example 3, even when glycerin was used as the plasticizer, a color reaction by the phosphatase substrate was confirmed.

  The microorganism culture sheet of the present invention can be used for, for example, detection of bacteria or inspection of the number of bacteria.

DESCRIPTION OF SYMBOLS 10 Base material sheet 20 Frame layer 30 Culture layer 30 'Specific component layer 40 Cover sheet 50 Adhesive layer 60 Double-sided adhesive tape

Claims (3)

A microorganism culture sheet having a culture layer,
The culture layer contains a chromogenic enzyme substrate and a plasticizer;
The chromogenic enzyme substrate is a chromogenic enzyme substrate that is degraded by phosphatase,
A microorganism culture sheet, wherein the plasticizer is glycerin.   The microorganism culture sheet according to claim 1, which is used for detecting Staphylococcus aureus. A method for detecting Staphylococcus aureus in a sample containing at least one kind of bacteria, wherein the culture layer of the microorganism culture sheet according to claim 1 or 2 is inoculated with at least a part of the sample. When,
Culturing bacteria inoculated on the culture layer;
Including methods.

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